Recombinant DNA Technology

Plasmids, Vectors, Cloning and Expression of genes

By,Abhinava J V

University of Agricultural Sciences, Dharwad

Vectors•Vector is an agent that can carry a DNA fragment into a host cell in which it is capable of replication.

• If it is used only for reproducing the DNA fragment, it is called a cloning vector.

•If it is used for expression of foreign gene, it is called an expression vector.

Properties of a good vector:•It should be autonomously replicating i.e. it

should have ori region.•It should contain at least one selectable

marker e. g. gene for antibiotic resistance.•It should have unique restriction enzyme

site (only one site for one RE) for different REs to insert foreign DNA.

•It should be preferably small in size for easy handling.

•It should have relaxed control of replication so that multiple copies can be obtained.

Types of vectors

•Plasmid vectors•Bacteriophage vectors•Cosmids •BACs & YACs•Mini chromosomes

Plasmids•Plasmids are extrachromosomal circular

DNA molecule that autonomously replicates inside the bacterial cell.

• Cloning limit: 100 to 10,000 base pairs or 0.1-10 kilobases (kb) .

• In their simplest form, plasmids contains a bacterial origin of replication, an antibiotic resistance gene, and at least one unique restriction enzyme recognition site which helps in cloning.

Advantages of Plasmids in Molecular Biology

•Easy to work with - Plasmids are a convenient size (generally 1,000-20,000 base pairs).

•Self-replicating - Endless number of copies of the plasmid was obtained by growing the plasmid in bacteria.

•Stable - Plasmids are stable long-term either as purified DNA or within bacteria (as glycerol stocks).

•Functional in many species and can useful for a diverse set of applications - Plasmids can drive gene expression in a wide variety of organisms, including plants, worms, mice and even cultured human cells.

Major Limitation of Cloning in Plasmids

Upper limit for clone DNA size is 12 kb Requires the preparation of “competent”

host cells Inefficient for generating genomic libraries

as overlapping regions needed to place in proper sequence

Preference for smaller clones to be transformed

If it is an expression vector there are often limitations regarding eukaryotic protein expression.

PUC 18/19•It is a circular double stranded DNA and

has 2686 base pairs and it includes: 1. A gene for antibiotic resistance to

Ampicillin (ampR).2. A gene (and its promoter) for the

enzyme beta-galactosidase (lacZ). 3. The lacZ gene contains a polylinker

region, with a series of unique restriction sites called as multiple cloning site (MCS) found in the plasmid.

pBR322•It is a circular double stranded DNA and has

4361 base pairs.•pBR322 also contains the ampR gene (source

plasmid RSF2124)• The tetR gene (source plasmid pSC101), •the rop gene encoding a restrictor of plasmid

copy number. •The plasmid has unique restriction sites for

more than 40 restriction enzymes. •11 of these 40 sites lie within the tetR gene. • There are 6 key restriction sites inside the

ampR gene

Bacteriophage Vector•Phage - derivatives of bacteriophage lambda;

linear DNA molecules, whose region can be replaced with foreign DNA without disrupting its life cycle; cloning limit: 8-20 kb

•Lambda viral genome: 48.5 kb linear DNA with a 12 base ssDNA "sticky end" at both ends; these ends are complementary in sequence and can hybridize to each other (this is the cos site: cohesive ends).

•Infection: lambda tail fibres adsorb to a cell surface receptor, the tail contracts, and the DNA is injected.

•The DNA circularizes at the cos site, and lambda begins its life cycle in the E. coli host.

Advantages:Useful for cloning

large DNA fragments

(10 - 23 kbp)Inherent size

selection for large inserts

Disadvantages:Less easy to handle

Cosmids •Cosmids - an extrachromosomal circular

DNA molecule that combines features of plasmids and phage; cloning limit - 35-50 kb

•Advantages:▫Useful for cloning very large DNA

fragments (32 - 47 kbp)▫Inherent size selection for large inserts▫Handle like plasmids

•Disadvantages:▫Not easy to handle very large plasmids ▫(~ 50 kbp)

BACs & YACs•Bacterial Artificial Chromosomes (BAC) - based

on bacterial mini-F plasmids. cloning limit: 75-300 kb •Yeast Artificial Chromosomes (YAC) - an artificial

chromosome that contains telomeres, origin of replication, a yeast centromere, & a selectable marker for identification in yeast cells; cloning limit: 100-1000 kb

•Advantages:▫Useful for cloning extremely large DNA fragments (100 - 2,000 kbp)▫This is very important for genome sequencing

projects•Disadvantages:

▫Not easy to handle extremely large DNA molecules

YAC vector

telomere telomerecentromereURA3ARS HIS3

replicationorigin

markers

largeinserts

Mini chromosomes•It is the next generation vector

used in Recombinant DNA Technology.

•A mini chromosome is a small chromatin-like structure consisting of centromeres, telomeres and replication origins and little additional genetic material.

•They replicate autonomously in the cell during cellular division.

•Mini chromosome vector having a cloning limit up to 10 Mb.

Cloning•Molecular cloning is a set of techniques used

to insert recombinant DNA from a prokaryotic or eukaryotic source into a replicating vehicle such as plasmids or viral vectors.

•Cloning refers to making numerous copies of a DNA fragment of interest, such as a gene.

•Cloning is the cutting a piece of DNA from one organism and inserting it into a vector where it can be replicated by a host organism.

Tools for cloning:

Restriction Enzymes

DNA ligase

Cloning Vectors

Restriction Enzymes•Restriction Enzymes (also called

Restriction Endonucleases) are proteins that cleave DNA molecules at specific sites, producing discrete fragments of DNA.

•Each enzyme recognizes and cuts specific DNA sequences.

•For example, BamHI recognizes the double stranded sequence:

5'--GGATCC--3'3'--CCTAGG--5'

Where do restriction enzymes come from?

•Restriction enzymes are found in bacteria. Bacteria use restriction enzymes to kill viruses – the enzymes attack the viral DNA and break it into useless fragments.

•They are named after the genus and species of the organism they were isolated from and are given a number to indicate the order in which they were found.

•For example, EcoRI was the first restriction enzyme isolated from Escherichia coli strain RY13, whereas HindIII was the third enzyme isolated from Haemophilus influenzae strain R d.

•Restriction enzymes cut through both nucleotide

strands, breaking the DNA into fragments, but they don’t always do this in the same way.

•Restriction enzyme that cuts straight through the DNA strands, creating DNA fragments with a flat or blunt end.

•Restriction enzymes, cut through the DNA strands at nucleotides that are not exactly opposite each other. This creates DNA fragments with one nucleotide strand that overhangs at the end. This overhanging nucleotide strand is called a sticky end because it can easily bond with complementary DNA fragments.

DNA ligase•DNA ligase (molecular glue)-Joins the two pieces of

DNA from different sources together through the formation of a covalent bond.

•Cells naturally carry out ligation during DNA replication, when the Okazaki fragments are joined together.

•Up to 1 million breaks can occur in the DNA of a single human cell each day.

•Ligases can join any DNA fragments with ‘blunt’ ends. They can also join DNA fragments with ‘sticky’ ends, but only if the nucleotides on the strands are complementary. To get complementary ‘sticky ends’ the DNA fragments to be joined must be cut with the same restriction enzyme.

Cloning Vectors•It should have unique restriction enzyme

site (only one site for one RE) for different REs to insert foreign DNA.

" RE

Cut plasmid vectorwith RE

RE RE

5´ 3´Excise DNA insert of interest from source using RE

Ligate the insert of interest into the cut plasmid

General Steps of Cloning with Any Vector

•Prepare the vector and DNA to be cloned by digestion with restriction enzymes to generate complementary ends.

• ligate the foreign DNA into the vector with the enzyme DNA ligase

•Introduce the DNA into bacterial cells (or yeast cells for YACs) by transformation

•Select cells containing foreign DNA by screening for selectable.

Methods for gene insertionTransformation Viruses based

Microinjection Agrobacterium

Screening of Recombinant Clones

Blue-White Screening

Expression of Gene•Expression cloning is a technique in DNA

cloning that uses expression vectors to generate a library of clones, with each clone expressing one protein.

•Gene expression involves: a) The production of messenger RNA by copying of the DNA template by RNA polymerase. b) Translation of the message into protein by the protein synthesis machinery.

•The transcriptional and translational signals may be synthetically created to make the expression of the gene of interest easier to regulate.

•After the insert of plasmid, it may not be able to generate functional protein from your cloned DNA.▫The gene may not be intact, or

mutations could have been introduced that disrupt it.

▫The protein encoded by the gene may require post-translational modifications (i.e., glycosylation or cleavage) to function.

▫Also, some enzymes are a complex of peptides expressed from separate genes.

promoter1 Cauliflower mosaic virus (CaMV)

2 opine promoters

3 plant ubiquitin (Ubi),

4 rice actin 1 (Act-1)

5 maize alcohol dehydrogenase 1 (Adh-1)